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Biomedical subjects

R A Proctor

Publications and source records attributed to R A Proctor.

At least 19 recordsLinked to original sources
PubMed1995

Persistent and relapsing infections associated with small-colony variants of Staphylococcus aureus.

Small-colony variants (SCVs) of Staphylococcus aureus were cultured from five patients with persistent and relapsing infections. All five SCV strains were nonhemolytic and nonpigmented and grew very slowly on routine culture media in an ambient atmosphere. In several instances, these phenotypic characteristics led to the initial misidentification of the organisms in the clinical microbiology laboratory. All four strains available for further analysis were shown to be auxotrophs that reverted to normal growth and morphology in the presence of menadione, hemin, and/or a CO2 supplement. Similarly, these isolates were resistant to aminoglycosides under routine conditions but susceptible in the presence of the metabolic supplements. For two patients, the large and small colony forms isolated concurrently were indistinguishable when analyzed by pulsed field gel electrophoresis and thus represented phenotypic variants within individual clones. We propose a model relating the phenotypic characteristics of S. aureus SCVs with the clinical pattern of persistent and relapsing infection.

Aged↗
PubMed1995

Use of adhesion-defective mutants of Staphylococcus aureus to define the role of specific plasma proteins in promoting bacterial adhesion to canine arteriovenous shunts.

We used an ex vivo canine arteriovenous shunt model, previously developed to study plasma protein adsorption and thrombogenesis on polymeric biomaterials, to define the role of host proteins in promoting adhesion of Staphylococcus aureus. Either polyethylene or polyvinyl chloride tubings were exposed to canine blood for 5, 15, or 60 min at a flow rate of 300 ml/min and then were flushed in phosphate-buffered saline (PBS), cut into 1.5-cm segments, and stored at -70 degrees C. After thawing, each segment was preincubated in 0.5% albumin in PBS to prevent nonspecific staphylococcal attachment to surfaces that were not exposed to blood. Each segment was then incubated with 4 x 10(6) CFU of [3H]thymidine-labelled S. aureus per ml for 60 min at 37 degrees C in an in vitro adhesion assay. Two site-specific mutants of S. aureus were tested: one specifically defective in adhesion to surface-bound fibronectin (FnAd-def) and the other defective in adhesion to fibrinogen (FgAD-def) [corrected]. Compared with their respective parental strains, the FgAd-def, but not the FnAd-def, mutant of S. aureus showed a strong (> 80%) decrease in attachment to ex vivo tubings. The adhesion of each strain of S. aureus onto polyethylene was consistently more than twofold higher than the adhesion onto polyvinyl chloride segments exposed to flowing blood for 5 or 15 min, but adhesion became similar to that on polyvinyl chloride after 60 min of exposure. In conclusion, the specific adhesion-defective mutants of S. aureus suggested that fibrinogen was the most active adhesion-promoting protein in a short-term blood-material interaction. The experimental approach described in this study should prove useful for screening materials thought to be resistant to protein-mediated staphylococcal adhesion and colonization.

Animals↗
PubMed1995

Quantitative comparison of clumping factor- and coagulase-mediated Staphylococcus aureus adhesion to surface-bound fibrinogen under flow.

The contributions of clumping factor and coagulase in mediating Staphylococcus aureus adhesion to surface-adsorbed fibrinogen have been quantified by using a new methodology and analysis. The attachment or detachment kinetics of bacteria were directly observed in a radial flow chamber with a well-defined laminar flow field and a spatially varying shear rate and were quantified by recursively scanning the chamber surface and counting cells via automated video microscopy and image analysis with a motorized stage and focus control. Intrinsic rate constants for attachment or detachment were estimated as functions of shear rate for the wild-type Newman strain of S. aureus and for mutants lacking clumping factor, coagulase, or both proteins on surfaces coated with plasma, fibrinogen, or albumin. Clumping factor, but not coagulase, increased the probability of attachment and decreased the probability of detachment of S. aureus on plasma-coated surfaces; however, both clumping factor and, to a lesser extent, coagulase increased the probability of attachment on the purified-fibrinogen-coated surface. All mutants were resistant to detachment on the purified-fibrinogen-coated surface, suggesting the possibility of an additional adhesion mechanism which was independent of coagulase or clumping factor and effective only for fully attached cells. Together, these results suggest that the presence of clumping factor plays the primary role in enhancing adhesion to surfaces with adsorbed fibrinogen, not only by enhancing the probability of cell attachment but also by increasing the strength of the resulting adhesion.

Bacterial Adhesion↗
PubMed1995

Alterations in clinical, hematological and metabolic variables in bovine neonatal endotoxemia.

Endotoxemia is an important cause of morbidity and mortality in the neonate. Although many models are used to study the problem, none completely simulates the natural disease. To more clearly define a bovine neonatal endotoxemia model we studied the effects of dose of endotoxin on clinical, hematological and biochemical variables. Thirty-four neonatal calves were administered Escherichia coli endotoxin (LPS) at 0 (0.9% saline solution), 0.2, 2.0 or 20 micrograms/kg, by either IV bolus or infusion over 50 minutes. Variables monitored included mean arterial blood pressure (MAP), leukocyte (WBC) count, plasma glucose and lactate concentrations and clinical status. All LPS-treated calves displayed similar clinical signs within one hour. Dose-dependent differences in response to LPS among groups became evident over time. Substantial dose-dependent changes in attitude, appetite, mucous membrane character, capillary refill time, MAP, plasma glucose and lactate concentrations, and WBC count were noted in LPS-treated calves. Higher doses of LPS induced a more prolonged clinical response and significantly (p < 0.05) greater hypotension, lacticemia and hypoglycemia. While dose altered the response to endotoxin, the method of administration had no overall effect on the variables measured.

Analysis of Variance↗
PubMed1994

Protection of mice from endotoxic death by 2-methylthio-ATP.

The lethal effects of endotoxin, a bacterial product shed into the blood during bacteremia, are thought to be due to macrophage release of mediators such as tumor necrosis factor alpha and interleukin 1. Although much is known about the pathophysiology of endotoxemia, relatively little is known about the cellular signaling mechanisms that are involved. The data in this study suggest that extracellular adenine nucleotides can influence the development of endotoxin shock. An adenine nucleotide analog, 2-methylthio-ATP, inhibited the endotoxin-stimulated release of toxic mediators (i.e., tumor necrosis factor alpha and interleukin 1), and it protected mice from endotoxin-induced death. These studies suggest a fundamental and unusual role for adenine nucleotides on endotoxin action, and they provide a potentially new therapeutic approach for the control of the pathophysiology of Gram-negative septicemia.

Adenosine Diphosphate↗
PubMed1994

Gentamicin-resistant menadione and hemin auxotrophic Staphylococcus aureus persist within cultured endothelial cells.

Staphylococcus aureus menadione and hemin auxotrophs, generated by in vitro gentamicin selection, demonstrated reduced hemolytic activity and enhanced intracellular survival within cultured bovine aortic endothelial cells relative to their hemolytic parent. Supplementation of the auxotrophs with exogenous menadione or hemin resulted in rapid growth, increased hemolytic activity, and reduced intracellular persistence to the level found for the hemolytic clinical parent. Aminoglycoside selection of staphylococcal menadione and hemin auxotrophs and subsequent persistence of these variants in the intracellular milieu may adapt S. aureus for evasion of host defenses and resistance to antimicrobial therapy.

Animals↗
PubMed1994

Variant subpopulations of Staphylococcus aureus as cause of persistent and recurrent infections.

While S. aureus small colony variants (SCVs) have been recognized in clinical materials for decades, only recently have these strains been linked to persistent, resistant, and relapsing infections. The biochemical basis for this phenotype appears to be reduced electron transport, which leads to many changes in these organisms, including decreased alpha-toxin production. Reduced alpha-toxin has been found to facilitate bacterial survival within cultured endothelial cells. This SCV subpopulation is more resistant to antibiotics, grows very slowly, and demonstrates unusual colony morphology. Hence, these resistant strains can be easily overloaded in the clinical microbiology laboratory, and may be resistant to conventional antibiotic therapy. Clinicians should ask the laboratory to search for SCVs with relapsing and resistant S. aureus infections.

Animals↗
PubMed1993

Effect of dose and method of administration of endotoxin on cell mediator release in neonatal calves.

The cellular response induced in the host animal by endotoxin contributes greatly to the morbidity and mortality of gram-negative infections in bovine neonates. We characterized the temporal sequence, magnitude, and duration of mediator release during endotoxemia and evaluated the effect of endotoxin dose and method of administration. Thromboxane B2 (TxB2), and 6-keto prostaglandin F1 alpha (PGF1 alpha) concentrations and tumor necrosis factor (TNF), and interleukin-1 beta (IL-1 beta) activities were measured in 34 newborn calves given Escherichia coli endotoxin at dosage of 0 (saline solution), 0.2, 2.0, or 20 micrograms/kg of body weight, either by IV administered bolus or infusion over 50 minutes. In all groups and at each lipopolysaccharide dosage, mediators peaked in this sequence: TxB2 and TNF, followed by PGF1 alpha, then IL-1 beta. Neither dose nor method of administration affected the sequence of mediator release. The magnitude of eicosanoid response to endotoxin was dose-dependent. During induced endotoxemia, duration and/or magnitude of mediator response reflected the dose of endotoxin administered, indicating that the outcome of endotoxemia, in neonatal calves, may be related to the amount of circulating endotoxin.

6-Ketoprostaglandin F1 alpha↗
PubMed1992

Clinical and biologic effects of combination therapy with gamma-interferon and tumor necrosis factor.

Tumor necrosis factor (TNF) and gamma-interferon (gamma-IFN) are cytokines with synergistic biologic and antiproliferative effects in vitro and in mouse models. The biologic effects of the combination of TNF and gamma-IFN, however, have not been studied well in humans. A Phase I trial was conducted of TNF and gamma-IFN therapy in 24 patients with advanced malignancies to determine the tolerability of the combination and the biologic effects of TNF and gamma-IFN in vivo. Both TNF and gamma-IFN were administered as 30-minute intravenous infusions three times per week. Doses of TNF ranged from 25 to 100 micrograms/m2; all patients received 100 micrograms/m2 of gamma-IFN. Dose-limiting toxicity consisted primarily of orthostatic hypotension and constitutional symptoms. The maximum tolerated dose level (MTDL) of 50 micrograms/m2 of TNF and 100 micrograms/m2 of IFN-gamma was less than the maximum tolerated dose (MTD) observed in previous Phase I trials of gamma-IFN and TNF alone. Biologic responses were studied in seven patients treated at the MTDL. Serum interleukin-2 receptor levels and neopterin secretion were enhanced significantly 24 hours after therapy (P = 0.002); enhancement of monocyte Fc receptor levels had borderline statistical significance (P = 0.07). With the exception of the mean fluorescent intensity on monocytes positive for histocompatibility antigen HLA-DR (P = 0.03), HLA Class I and II cell surface protein expression was not increased. The combination significantly enhanced indoleamine dioxygenase activity and serum beta 2-microglobulin expression (P less than 0.04) but not 2',5'-oligoadenylate synthetase activity, bactericidal function, or chemiluminescence. These results were compared retrospectively with those observed in previous Phase I trials of gamma-IFN and TNF alone. The combination of TNF and gamma-IFN significantly increased urinary kynurenine levels more than either TNF alone or gamma-IFN alone. Given the limitations inherent in any retrospective analysis, however, the enhancement in the other biologic parameters measured at the MTDL during this trial did not differ significantly from the changes observed at the MTD of either TNF or gamma-IFN alone. It was concluded that the combination of TNF and gamma-IFN, when administered at the MTDL of the combination, does not offer any enhancement in biologic responses over either agent alone.

2',5'-Oligoadenylate Synthetase↗
PubMed1991

Bacterial lipopolysaccharide-stimulated GTPase activity in RAW 264.7 macrophage membranes.

The molecular mechanisms surrounding the toxicity and high mortality rate that accompany the release of bacterial lipopolysaccharide (LPS) are unclear, although its potent activity suggests that an amplification system is involved. Because previous studies suggest that a guanine-nucleotide-binding protein (G-protein) may participate in LPS action, we have evaluated the effects of LPS on GTPase activity in membranes isolated from macrophage (RAW 264.7) and fibroblast (B82L) cell lines. LPS induced substantial GTPase activation (200-300% above basal), and kinetic analyses indicated that the maximal LPS-stimulated increase in velocity is observed within 15 min, that it is a low-Km (for GTP) activity, that it can be enhanced by ammonium sulphate, and that it appears to be pertussis toxin-insensitive. Moreover, the LPS-enhanced GTPase activity was not antagonized by phosphatase/ATPase inhibitors such as p-nitrophenyl phosphate, ouabain, bafilomycin or N-ethylmaleimide, and in fact was potentiated by the addition of ATP or ADP. Conversely, the LPS precursor, lipid X, which can decrease the lethal effects of LPS, was found to dose-dependently inhibit the LPS-mediated stimulation of GTPase activity. Half-maximal inhibition was seen at the same lipid X/LPS ratio known to be effective in vivo, i.e. 1:1(w/w). These effects appear to be specific because other phospholipids, detergents and glycosides neither stimulated basal, nor inhibited LPS-induced, GTPase activity. These data suggest the involvement of a GTPase in LPS action, and indicate that lipid X may act to directly antagonize LPS at this level.

Adenosine Triphosphate↗
PubMed1991

Biological and clinical effects of intravenous tumor necrosis factor-alpha administered three times weekly.

Tumor necrosis factor (TNF) is a cytokine with pleiotropic biological and antitumor effects in vitro and in mouse models. The immunological effects of the molecule as a single agent, however, have not been well studied clinically. We conducted a Phase I trial of TNF in 53 patients with advanced malignancies in order to determine the biological and clinical effects of TNF when administered as a 30-min i.v. infusion three times/week. Dose levels of TNF ranged from 5 to 275 micrograms/m2; doses of TNF were escalated between patient groups. The most common clinical toxicities of TNF consisted of rigors, anorexia, headache, and fatigue. Dose-limiting toxicity consisted of hypotension, fatigue, and nausea. Four patients treated at the maximally tolerated dose of 225 micrograms/m2 received dexamethasone to determine whether the toxicities of TNF could be ameliorated. No significant differences in hypotension or subjective symptomatology were observed in those patients receiving dexamethasone and those who did not or between injections in which dexamethasone was administered and when it was not. One patient with colorectal carcinoma treated with 50 micrograms/m2 had a partial response lasting about 9 months. Biological responses were evaluated in 8 patients treated at the maximally tolerated dose before therapy and 24 h afterward. TNF significantly (P less than 0.05 for all) enhanced serum beta 2-microglobulin, serum neopterin, and serum interleukin-2 receptor (Tac antigen) levels. Indoleamine 2,3-dioxygenase activity was also increased 24 h following the administration of TNF, although this increase was only of borderline statistical significance (P = 0.07). TNF did not enhance granulocyte bactericidal activity. The expression of cell surface proteins on monocytes, including HLA-DR, HLA-DQ, beta 2-microglobulin, and the Fc receptor, and serum interleukin-1 activity also were not significantly increased by the administration of TNF. Thus, in humans TNF caused biological response modulation with evidence of HLA Class I (beta 2-microglobulin) increase and T-cell (Tac antigen) and monocyte (neopterin) activation.

2',5'-Oligoadenylate Synthetase↗
PubMed1991

Immunostimulatory, but not antiendotoxin, activity of lipid X is due to small amounts of contaminating N,O-acylated disaccharide-1-phosphate: in vitro and in vivo reevaluation of the biological activity of synthetic lipid X.

Lipid X, a precursor in the biosynthesis of lipid A, has been claimed to possess most of the immunostimulatory activity but none of the toxicity of endotoxin. However, recent work shows that synthetic lipid X can be contaminated with small amounts of N,O-acylated disaccharide-1-phosphate (H. Aschauer, A. Grob, J. Hildebrandt, E. Schuetze, and P. Steutz, J. Biol. Chem. 265:9159-9164, 1990). Because the impurities themselves exhibit immunostimulatory properties, it was necessary to establish whether chemically pure synthetic lipid X exhibits any of the endotoxinlike biological activities previously attributed to the native compound extracted from the Escherichia coli MN7 mutant. In the present study, two batches of synthetic lipid X were used: batch A contained the contaminating disaccharide, and batch B was pure lipid X. Batch A, previously believed to be pure on the basis of spectroscopic and microanalysis data, induced murine peritoneal macrophages to secrete tumor necrosis factor, interleukin-1, and prostaglandin E2 at a minimal dose of 10 micrograms/ml in vitro. Batch B, further purified by Sephadex LH 20 chromatography, was found virtually inactive in these in vitro assays. Furthermore, batch A was pyrogenic in rabbits at a dose of 0.05 mg/kg, whereas batch B was not pyrogenic at doses of up to greater than or equal to 2 mg/kg. However, both batches were equally tolerated by galactosamine-loaded mice at doses of up to 100 mg/kg. Surprisingly, while only batch A protected neutropenic mice against lethal infection with Pseudomonas aeruginosa (50% effective dose, 12.4 mg/kg), both batches were equally protective against infection with herpes simplex virus type 1 in mice and guinea pigs, even when lipid X was administered therapeutically. Interestingly, both batches of lipid X blocked endotoxin-induced expression of monocyte procoagulant activity and priming of human neutrophils for superoxide anion release. Similarly, both batches protected galactosamine-sensitized mice from otherwise lethal endotoxemia when administered prophylactically or simultaneously with the lipopolysaccharide challenge. Thus, our findings suggest that chemically pure lipid X (batch B) is devoid of the immunostimulatory properties of lipid A or endotoxin. Rather, it behaves as a competitive inhibitor of lipopolysaccharide. We conclude, therefore, that previous studies which attributed immunostimulatory activities to any batch of synthetic lipid X should be interpreted with caution.

Adjuvants, Immunologic↗
PubMed1990

Lipid A precursors protect against endotoxin challenge.

These studies provide exciting prospects for the future treatment of gram-negative infections. The anti-endotoxin activity of lipid X, a monosaccharide precursor of lipid A, may be a prototypic compound for agents that can block the toxic effects of endotoxin that is being actively released. In contrast, monophosphoryl lipid A, a disaccharide derivative of lipid A, stimulates host defenses against infections and tumors. With further understanding of the mechanisms by which these compounds exert these effects, we can anticipate that new and more active compounds will be developed and that further activities of existing compounds will be found.

Animals↗